The general goal of this study is to examine gene expression during embryonal carcinoma differentiation and to try to understand the differential regulation of genes during that differentiation. We have synthesized single-stranded (ss) and then double-stranded (ds) cDNA to PFHR-9 endodermal polyadenylated, cytoplasmic RNA. The ss cDNA has an average size of approximately 1,400 bases, and the ds cDNA has an average size of 800 base pairs. This ds cDNA has been tailed with deoxycytidine (dC) using terminal transferase and inserted in the vector pBR322, which has been linearized at the Pst I site and tailed with (dG). A small cDNA library containing over 1,000 independent colonies has been isolated and we are now examining it for: (1)\average insert size; and (2)\whether the inserts are for genes expressed only in the endodermal cells or are expressed in both the stem cell [embryonal carcinoma (EC)] and the endodermal cells. This is preliminarily being examined by hybridization of H3-uridine labeled RNA from either F9 EC cells or PRHR-9 endodermal cells to selected recombinants and to small pools of recombinants. The final development has involved the use of synthetic oligonucleotides to obtain specific cDNA recombinants. Certain sequences in this region are important for the expression of the HSVtk gene. We plan to use these recombinants to see if we can select, from F9tk(-) cells, colonies which are resistant to hypoxanthine-aminopterin-thymidine medium after transfecting the cells with these recombinants. A cDNA library was constructed from human RNA using as a primer a set of oligonucleotides representing a specific amino acid sequence of a human gene. A recombinant was isolated having an insert which hybridizes to the primer and also to an oligonucleotide of a set representing an amino acid sequence over 100 base pairs upstream from the primer oligonucleotide. Along with sequencing this cDNA insert, we plan to insert it into the above-described recombinants to discover whether it might be expressed in either bacteria or transfected EC cells. A positive result from this approach would suggest that it would be feasible to cDNA clone the laminin molecules once amino acid sequencing becomes available.